The molecular parameters of tryptophan hydroxylase from rat brainstem and murine mast cell, were determined by gel filtration and sucrose density gradient ultracentrifugation. The enzyme from rat brainstems has a calculated molecular weight of 220,000 daltons, a Stokes radius of 55.6A, a frictional ratio of 1.28, and a sedimentation coefficient of 9.63S. The mast cell enzyme has a molecular weight of 144,000 daltons, a Stokes radius of 50.3A, a frictional ratio of 1.35, and a sedimentation coefficient of 6.97S. Evidence for catalytically active subunits was not found. The regulatory and kinetic properties of the brainstem and mast cell hydroxylase were also compared. The brain enzyme can be activated by calcium, SDS, trypsin, phospholipids, protein phosphorylation, and by heparin. Of these treatments only heparin activated the mast cell tryptophan hydroxylase. It appears that the tryptophan hydroxylase species form rat brainstem and murine mast cell represent distinct molecular entities.